The structure of the oxysterol binding protein and the kinetics of 25-hydroxycholesterol binding were shown to be markedly affected by several factors. The molecular weight of the unliganded binding protein at pH 7.4 is estimated to be approximately 220,000 (calculated from measurements of the sedimentation coefficient and the Stokes radius. Binding of the diol is relatively slow at pH 7.4--48 hours was required to reach equilibrium. Alteration of the pH to 6 or lower results in a decrease in the apparent Mr to about 163,000, and the rate of binding of the ligand is greatly increased--about 1.5 hours was required to reach equilibrium. After exposure to acid pH (pH 5.5) in the presence of 2.5 M urea the protein-sterol complex is further dissociated to a lower Mr form, about 90,000. The dissociation constant (Kd = 10-9 M) for the sterol-protein complex is relatively unaffected by changes in the structure of the protein. The physiological significance of these observations is unknown, but they are reminiscent of alterations in steroid hormone receptors associated with transformation to a nuclear binding form. Considerable support for the hypothesis that the binding protein is involved in the regulation of HMG-CoA reductase was obtained by comparing the apparent affinities of 36 different sterols for the binding protein with their abilities to repress the synthesis of HMG-CoA reductase in cell cultures. Over a 100-fold range of activity was seen in one test; activity in the other was directly proportional. We have used the binding protein to detect a "regulatory" oxysterol in cultured cells. The "regulatory" sterol was isolated by HPLC and was shown to repress HMG-CoA reductase in cultured cells. Its identity is not yet established.